Conventionally, an exposed conductive region having a micrometer scale or larger area can be formed on an insulated metallic substrate by means of a variety of techniques pertaining to the well-known semiconductor process. Due to the limitations of conventional techniques, however, a formation of exposed conductive regions of a nanometer scale faces a number of obstacles. Various approaches have been attempted to overcome these obstacles. See, for example, U.S. Pat. Nos. 6,503,701, 6,492,096, 6,322,963, 6,001,587, and 5,922,2146.
On the other hand, a monolayer of self-assembled molecular (hereinafter, referred to a “monolayer SAM”) may be described by a state in which an organic compound containing thiols (—SH) or disulfides (—S—S) is aligned in the form of a mono-layer by covalent-bonding of the sulfuric functional group of the compound with a metallic electrode made of gold or platinum or the like. For example, the monolayer SAM may be formed by steps of preparing an aqueous solution of the self-assembling material dissolved in a solvent such as water or various organic solvent, contacting the solution with a metallic substrate (immersion in the solution or dropping of the solution on the substrate) and reacting for a certain period of time, and rinsing out the un-reacted reagents. These monolayer SAMs may be employed to modify a metallic surface, i.e., its hydrophobic surface to a hydrophilic, and vice versa. They also may serve as a linker for a bio-material to be bound to a metallic material. Although a number of patents have attempted to apply the SAM techniques to the fabrication of biosensors, it is found out that a commercialized biosensor using the SAM technique has not been reported yet.
The reported conventional techniques pertaining to the monolayer SAM are related mostly to methods of forming a monolayer SAM on a conductive metallic material such as gold or platinum or the like, techniques for fixing a bio-material, such as protein and DNA, to the functional groups in the end of the created SAM, and characterization of materials to be fixed to the SAM monolayer. Those techniques are disclosed, for example, in U.S. Pat. Nos. 4,964,972, 5,652,059, 5,686,549, 5,725,788, 5,827,417, 5,922,214, 6,031,756, 6,114,099, 6,127,127, 6,156,393, 6,183,815, 6,270,946, 6,287,874, 6,312,809, 6,322,979, 6,346,387, 6,432,723, 6,436,699, 6,444,318, 6,444,321, 6,492,096, and 6,114,099.
The quantitative analysis of protein has been carried out through the application of immuno-reaction, a typical one of which is the enzyme-linked immuno Sorbent Assay (ELISA). In ELISA, a primary antibody, which is specifically fixed to the protein to be analyzed, is fixed to a substrate. Thereafter, a secondary antibody bondable to the primary antibody is bound thereto. A label substance is attached to the secondary antibody and the label substance has fluorescence or radioactivity. Therefore, the concentration of the protein can be quantitatively analyzed by measuring the intensity of the signal resulting from the fluorescence or radioactivity.
It has been found out that the above conventional techniques have several problems. Typically, it requires a rinsing and cleaning procedure in order to remove non-specifically bonded enzyme or antibody, and needs separate equipment for detecting the fluorescence or radioactivity from the label substance. Those disadvantages lead to a large scale of the whole measurement outfit, a longer period of time for analyzing (for example, several hours), and an increase in the cost of equipment (for example, several tens of thousand dollars). There is therefore a limitation in commercializing a portable analyzer that can be used by small-sized hospitals or non-professional individuals.